Parathyroid hormone (PTH) is a polypeptide hormone which is naturally produced in the parathyroid of mammals. The human polypeptide consists of 84 amino acids and is involved in the regulation of the calcium concentration in blood plasma. If the calcium level drops below a threshold level, PTH is secreted by cells of the parathyroid glands into the blood and induces a release of calcium from bone tissue. At the same time, PTH supports calcium absorption from the small intestine and enhances calcium reabsorption from the primary urine, thereby suppressing calcium loss via the kidneys. Due to its calcium releasing effects, an excessive amount of PTH in the blood as commonly observed in primary and secondary hyperparathyroidism has been found to be associated with reduced bone density and bone atrophy (osteoporosis). Considering the physiological effects of PTH, it appears odd that the hormone nevertheless proved useful in the therapy of osteoporosis.
However, animal studies in rats for the first time revealed that a short-term exposure to PTH supports bone formation due to transient activation of osteoclasts, whereas a sustained exposure ultimately results in bone atrophy. Subsequent clinical studies in humans using the pharmaceutically active fragment PTH1-34 of human PTH confirmed that the fragment can be used for treating osteoporosis. PTH1-34 is a polypeptide having a molecular mass of 4.7 kDa that consists of the first 34 amino acids of the human PTH hormone. It was approved for osteoporosis therapy in 2002 under the product name “teriparatide”. Teriparatide is sold by Lilly Pharma under the trade name Forteo (in the US) and Forsteo (in Europe).
At present, PTH1-34 for therapeutic use is produced by heterologous expression in bacterial host cells and subsequent purification of the pharmacological active polypeptide. However, it has been found that all known fragments and variants of PTH1-34 occur during the production process demonstrate a reduced potency compared to PTH1-34. In addition a negative effect on overall purity is observed. For example, a number of fragments derived from PTH1-34 were identified in the final batch obtained after heterologous expression, including amongst others PTH1-30, PTH2-34, PTH3-34 and PTH4-34. Moreover, chemical modifications of certain amino acids of PTH1-34, e.g. oxidation of methionine residues or deamidation of asparagine residues, give rise to additional variants.
However, as homogeneity of a product intended to be used as a therapeutic is of utmost importance for safety reasons, any kind of truncated or chemically modified form of PTH1-34 in the final product is clearly undesirable. Accordingly, current production processes normally include one or more chromatography steps that aim at the purification of the PTH1-34 polypeptide. However, it has been found to be problematic to separate PTH1-34 from some of its variants and fragments without significant product loss. In particular, it was observed that the fragment PTH2-34 shows essentially the same retention properties in ion exchange chromatography as PTH1-34. Consequently, the PTH2-34 fragment is co-eluted with PTH1-34 which makes PTH1-34 purification challenging. Moreover, alternative chromatographic techniques, e.g. reversed phase chromatography, were tested but did not show any potential for a preparative separation of truncated and full-length teriparatide.
In light of the above, new methods are needed which are effective in separating PTH1-34 from its fragments and chemically modified variants. The method of the present invention provides for the effective purification of the PTH1-34 polypeptide by cation exchange chromatography using inversely directed gradients of pH and ionic strength. As discussed in more detail below, the method is particularly suitable for separating PTH1-34 from PTH2-34.